We started studying the amyloids in the form of yeast prion structures in 1998, focussing initially on Ure2p, a negative regulator of nitrogen catabolism. We showed that its N-terminal domain is responsible for prionogenesis, while the C-terminal domain which performs its regulatory function remains folded in filaments but is inactivated by a steric mechanism. In our amyloid backbone concept, the prion domains form the filament backbone and are surrounded by the C-terminal domains. In 2005, we published the parallel superpleated beta-structure model for the amyloid backbone. It envisages arrays of parallel beta-sheets generated by stacking monomers with planar beta-serpentine folds. Topologically similar structures are good candidates for other amyloid fibrils, including amylin and growing support for models of this kind is appearing in the scientific literature. Our ongoing work is aimed at testing and refining this model;exploring its range of applicability;and investigating fibril polymorphism. In FY10, we focussed on three areas: (1) Systematic analysis of beta-arcade motifs in amyloid fibril models and beta-solenoid protein structures. Amyloid fibrils accumulate in diseases, such as Alzheimers or type II diabetes. The amyloid-forming protein is disease-specific. Amyloids may also be formed in vitro from many other proteins. Unlike the diverse native folds of these proteins, their amyloids are fundamentally similar in being rigid, smooth-sided, and in having cross-beta structures. Despite the difficulties attendant upon obtaining high resolution experimentally determined fibril structures, increasingly credible models are being derived by integrating data from multiple techniques. Most current models of disease-related amyloids invoke beta-arcades, columnar structures produced by in-register stacking of beta-arches. A beta-arch is a strand-turn-strand motif in which the two beta-strands interact via their side-chains, not via the polypeptide backbone as in a conventional beta;-hairpin. Crystal structures of beta-solenoids, a class of proteins with amyloid-like properties, offer insight into the beta-arc turns found in arches. General thermodynamic considerations suggest that complexes of two or more beta-arches may nucleate amyloid fibrillogenesis. (2) Formation of both infectious and non-infectious amyloid fibrils by the HET-s prion protein of the filamentous fungus, Podospora anserina. This prion differs from the yeast prions protein, Ure2p and Sup35p, in producing a gain-of-function prion, and in not having an abnormally high content of Asn/Gln residues. In 2007, we published a paper showing by electron diffraction that Het-s fibrils have a cross-beta structure (as anticipated), and by EM-based mass measurements that it has an axial packing density of 1 subunit per 0.94 nm - half the density of Ure2p and Sup35p fibrils, and in agreement with a published model, the stacked beta-solenoid. In another study, also completed in 2007, we compared the amyloids formed by the HET-s prion domain at different pH's. Fibrils formed at pH 7 from those formed at pH 2 on morphological grounds and in having higher specific infectivity. In our most recent line of investigation, we have explored the correlation between infectivity and fibril morphology further by cryo-EM. Above pH 3, singlet fibrils are produced while, below pH 3, triplet fibrils are obtained. Singlets have an axial periodicity of 40 nm and a left-handed twist. Triplet fibrils have three supercoiled protofibrils whose diameter (5 nm ), and axial packing density (1 subunit per 9.4 nm) resemble those of singlet fibrils but whose axial repeat and supercoiling differ. In triplet fibrils formed at higher pH, the interactions among protofibrils appear to loosen, eventually leading to their separation into individual protofibrils. Based on these observations, we have proposed that infectivity is based on the templating of "daughter" fibrils on the lateral surfaces of pre-existing "mother" fibrils and the loss of infectivity in triplet fibrils is caused by the occlusion of their lateral surfaces. (3) Manifestation of amyloid fibril backbone architecture in Sup35p prion filaments. In yeast cells infected with the PSI+ prion, the protein Sup35p forms aggregates and its activity in translation termination is down-regulated. Sup35p has an N-terminal prion domain;a highly charged middle (M-)domain;and a functional C-terminal domain. By negative staining, cryo-EM, and scanning transmission EM (STEM), in vitro-assembled filaments of full-length Sup35p show a thin backbone fibril surrounded by a diffuse cloud of C-domains, giving a full diameter of 65nm. In diameter (8 nm) and appearance, the backbones resemble amyloid fibrils of N-domains alone. STEM mass-per-unit-length data yield 1 subunit per 0.47 nm for N-fibrils, NM-fibrils, and Sup35p filaments, further supporting the amyloid fibril backbone model. The 30nm radial span of decorating C-domains indicates that the M-domains assume highly extended conformations. The extended M-domain conformations offer an explanation for residual Sup35p activity in infected cells, whereby the C-domains remain free enough to be capable of some interaction with ribosomes.